OPTIMIZATION OF PRIMER SEQUENCES FOR MOUSE SCFV REPERTOIRE DISPLAY LIBRARY CONSTRUCTION
OPTIMIZATION OF PRIMER SEQUENCES FOR MOUSE SCFV REPERTOIRE DISPLAY LIBRARY CONSTRUCTION
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DOI：
10.1093/nar/22.5.888
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发表时间：
1994-03-11
影响因子：
14.9
通讯作者：
PAPAS, TS
中科院分区：
文献类型：
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作者：
ZHOU, H;FISHER, RJ;PAPAS, TS
在免疫球蛋白库克隆中，引物序列与靶模板序列的同源性和引物库的多样性是决定克隆效率和文库大小的两个最重要的参数。在本报告中，我们提出了一组优化的引物，与商业上可获得的载体相容，用于小鼠scFv库噬菌体展示文库的构建。我们使用INMUNO-ZAP Fab克隆系统（1）在初步的努力中克隆单克隆杂交瘤，用于通过细胞内靶向对人原癌基因ETS 1和ETS 2的各个结构域进行功能表征。然而，在我们的手中，Immuno-ZAP相容引物仅在U-244（ETS 2特异性MAb）（2）的重链和T7（ETS 2特异性MAb）（2）和E44（ETS 1特异性MAb）（3）的轻链的PCR克隆中有效，而不是它们的伴侣链。这使我们尝试了一种scFv克隆系统（4），该系统已被证明在构建人天然抗体库展示文库中是有效的（5）。为了开发一组用于小鼠抗体库展示文库构建的相容性引物，我们汇集并排列了Kabat-Wu数据手册中收集的小鼠抗体序列（6）。将相似的序列分组，并从每组中提取推定的引物序列。然后借助于VAX计算机将所有推定的引物序列与所有汇集的序列进行比较，并选择最佳拟合的引物序列。重复该过程，直到覆盖所有序列。最后将与pHENI噬菌粒载体（4）相容的侧翼序列和接头（参见表1的注释）添加到VH和VL引物中，使得克隆的VH或VL片段可用于初级库克隆或通过链改组的亲和力改善（7）。计算机辅助序列同源性分析表明，10个产生的MHV。VH库克隆5 '引物在100%同源性下覆盖147个列出的小鼠重链序列中的55%，在> 95%时覆盖84%，在> 90%时覆盖97%，在> 86%时覆盖99%。相比之下，来自Immuno-ZAP系统（1）的8个5 '引物（HI-H8）仅覆盖所列小鼠VH序列的58.5%，同源性水平等于或大于82%。九个结果MKV。BACK VL κ链特异性5 '引物在100%同源性下覆盖67%的所列序列，在> 95%处覆盖84%，在> 90%处覆盖91%，并且在> 86%处覆盖99%。我们也
In immunoglobulin repertoire library cloning, the homology between a particular primer sequence and that of its target template, and the diversity capacity ofa primer pool are the two most important parameters whichdetermine the cloning efficiency and the size of a resulting repertoire library. In this report we present an optimized set of primers, compatible with a commercially available vector, for mouse scFv repertoire phage display library construction.We used the INMUNO-ZAP Fabcloning system (1) in an initial effort to clone monoclonal hybridomas for functional characterizations of the various domains of human protooncogene ETS1 and ETS2by intracellular targeting. In our hands, however, the Immuno-ZAP compatible primers were only effective in PCR-cloning of the heavy chain of U-244 (an ETS2-specific MAb)(2) and the light chains of T7 (an ETS2-specific MAb)(2) and E44 (an ETS 1-specific MAb)(3), but not their partner chains. This led us to try a scFv cloning system (4), which had been demonstrated effective in the construction ofa human naive antibody repertoire display library (5). To develop a set ofcompatible primers for mouse antibody repertoire display library construction, we pooled and lined up the mouse antibody sequences collected in the Kabat-Wu data book (6). Similar sequences were grouped and a putative primer sequence was drawn from each group. All the putative primer sequences were then each compared against all the pooled sequences with theaid ofa VAX computer and the best-fit primer sequences were selected. This process was repeated until all the sequences were covered. Flanking sequences compatible with the pHENI phagemid vector (4) and a linker (see notes for Table 1) were finally added to the VH and VLprimers so that cloned VH or VL fragments can be used either in primary repertoire cloning or in affinity improvement by chain shuffling (7). Computer-aided sequence homology analysis showed that the ten resulting MHV. BACK VH repertoire-cloning 5'primers cover 55% of the 147 listed mouse heavy chain sequences at 100% homology, 84% at> 95%, 97% at> 90% and 99% at> 86%. In contrast, the eight 5'primers (HI-H8) from Immuno-ZAP system (1) only cover 58.5% of the listed mouse VH sequences at a homology level equal or greater than 82%. The nine resulting MKV. BACK VL kappa chain specific 5'primers cover 67% listed sequences at 100% homology, 84% at> 95%, 91% at> 90% and 99% at> 86%. We also